AUTONOMOUS UNIVERSITY OF MADRID BIOCHEMISTRY DEPARTMENT Analysis of haploinsufficiency in women carrying germline mutations in the BRCA1 gene. Different mutations, different phenotypes? Tereza Vaclová MADRID, 2014 1 Cover design by Jiřina Vaclová Press financed by Human Cancer Genetics Programme (CNIO) 2 BIOCHEMISTRY DEPARTMENT FACULTY OF MEDICINE AUTONOMOUS UNIVERSITY OF MADRID Analysis of haploinsufficiency in women carrying germline mutations in the BRCA1 gene. Different mutations, different phenotypes? Doctoral thesis of M.Sc. in Molecular Biology and Genetics Tereza Vaclová Thesis directors Dr. Javier Benítez Ortiz Dr. Ana Osorio HUMAN GENETICS GROUP HUMAN CANCER GENETICS PROGRAMME SPANISH NATIONAL CANCER RESEARCH CENTRE 3 This thesis, submitted for the degree of Doctor of Philosophy at the Autonomous University of Madrid, has been elaborated in the Human Cancer Genetics laboratory at the Spanish National Cancer Research Center (CNIO), under the supervision of Dr. Ana Osorio and Dr. Javier Benítez Ortiz. This research was supported by following grants and fellowships: - La Caixa/CNIO International PhD Fellowship, 2010-2014: Tereza Vaclová - EMBO Short-Term Travel Fellowship, 2013: Tereza Vaclová - La Caixa/CNIO Short-Term Stay Fellowship, 2013: Tereza Vaclová - Spanish Ministry of Economy and Competitiveness (MINECO; SAF2010-20493) - Spanish Network on Rare Diseases (CIBERER) 9 This thesis is dedicated to my parents for their love, endless support and encouragement ♥ 11 12 ACKNOWLEDGEMENTS 14 This work would not be possible without a huge number of amazing people that I met during this four year roller coaster and it is my pleasure to have a chance to acknowledge them here. First of all, I would like to thank to my supervisors Javier and Ana, who modulated my scientific career the most and guided me throughout those four years. I am very thankful I had the opportunity to become a part of the Human Genetics lab, experience such friendly atmosphere there and learn so much from all the lab members. I am honestly so grateful for the way both of you mentored me and for the freedom and trust you showed me, because it taught me how to act responsibly and think critically about my work. Javier, I very much appreciate all your encouragements and positive attitude which kept the everyday stress at very low levels and created comfortable and relaxed working environment. I am grateful for all your comments and constructive criticism which contributed to my improvement! Ana, I admire your ability to be a teacher and a great friend at the same time. I can’t describe how much I am happy I had the pleasure to work under your supervision and get to know you also as a person. Thank you for all your advice, for the amount of time you dedicated to my work, for your kind support, interesting conversations about science and also about life. I sincerely can’t imagine better supervisors than you two, thank you for everything! I would also like to thank to my thesis committee members: Anna González Neira, Miguel Ángel Quintela and Javier Santos for our valuable discussions and comments, their support and feedback that they have been giving me. Thanks to all CNIO Units for their professionalism, suggestions and kind behavior. I would like to especially thank to members of the Confocal Unit, Diego, Manu and Joaquim, for helping me to take cool pictures and patiently answering all my questions. In addition, I am grateful for all the help I got from Guada and Puri from the Genomics Unit and for the help and suggestions related to expression data analysis that I got from Gonzalo from the Bioinformatics Unit. Importantly, non of this work would be possible without all the women who participated in the study and the help of Fernando Setién from Barcelona, who took care of the cell immortalization. During these more than four years, many people were part of the Human Genetics family and I am grateful I had a chance to meet each one of them. Dear Mili and Martička, I felt your friendship from the very first day and can’t describe how much I appreciate all the things you both did for me, in both good and bad times! Thank you for all the hours and hours of conversation, for your advice, help and support with my experiments. I will never forget all the fun and crazy jokes I experienced with you, countless talks about life and everyday troubles, and drinking litres and litres of café con leche :-) You became part of my new family here and I can’t wait for our reunion! Martičkovatět, “dzienki bardzo” for being an amazing “desk neighbor” and sharing your polish culture (and serek homogenizowany) with me :-) I am really grateful for all you ever did for me and for the jokes that made our working time so enjoyable! Kira, I really appreciate how nicely you treated me from the very beginning and and how optimistic person you have always been! I am happy I had a chance to get to know Ricardo and your parents, and I will never forget the relaxed atmosphere with you and your family and the crazy run in costumes on the New Year’s Eve 2012! Similarly, thank you so much, Nere, for your friendliness, kindness and all the fun we had together in the lab. I am really happy you shared the magic atmosphere of your pueblo Muñoveros with me and taught me the local “sombrero” 15 style of life :-) I can’t wait to take part at your thesis defense soon, we are both almost there ;-) Special acknowledgements to Ale, who quickly became my friend and shared her sensitive spirit with me. Thank you for your thoughts, ideas, all the interesting talks we had together, and also for rescuing me many times from the battle of thesis writing in the CNIO library :-) Ali, muchas gracias por ser siempre tan positiva y por toda tu ayuda con mis pedidos y otras cosas, nunca olvidaré tus bromitas en estilo “Genética Romana” y tu risa específica! I very much appreciate all the support, opinions and suggestions I received from María, both in relation to my experiments and to other issues, thank you so much for your kindness! I am also grateful to Carlos, who was always very kind and friendly and created so positive atmosphere in the lab with his endless jokes. Many thanks to Javi for being always very nice to me and for all the discussions related to cell culture and western-blot mysteries. Thanks to Toya for her kind support and help with tens of orders I made. I am also grateful to Fatima for her sense of humor and her ability to make a whole lab crying from laughing every day, to Oriol for being very nice and funny, Bea for her naturalness and kindness, to Samu for his help with statistical issues :-) I would also like to thank to Miguel for being extremely nice all the time. For sure, I can’t forget about all the former members of the lab, I thank to Bea for being always a positive person and giving me many advice related to my work. I also learned a lot from Laura, who took care of me at the beginning of my PhD, taught me a lot of cell culture tricks and was always very patient and helpful. Thank you LauPau for all your help, I appreciate it very much! Thanks to Maika for her graciousness and optimism which always made me smile. I also am happy I had a chance to meet Barbara, Eva, Ivan, Javi, Fer and all the inspiring students who spend some time in our lab: Laila, Natalia, Sergio, Sofia. Special thanks to my very smart summer student Sergio! I admire your positive attitude and all our experiment-related and -unrelated chats :-) It has been a pleasure for me to share some of my scientific skills with you, thank you for being so kind and inspirational! I can’t forget about people from other labs of the Human Cancer Genetics Programme, among which I made some very good friends. First of all, I thank to all current and former members of the Hereditary Endocrine Cancer group for the valuable discussions we had together in lab meetings and also out of the institute. Thanks to Meme, Cristina and Alberto for their constructive advice and comments and their kindness. I am also very thankful to Javi for his continuous smile and friendly behavior which helped me to overcome some crazy times with experiments. I also appreciate the kindness of Iñaki and had a lot of fun with our never-ending Czech/Spanish sport battles :-) Many thanks also to Lara, Alvaro, Maria A, Maria C, Rocio and Lucia for lending me a lot of their chemicals and being supernice to me all the time. The most special acknowledgements from this group belong to Vero... Vero, ani si neumíš představit (a nebo možná trošku umíš :-) ), jak moc jsem ti vděčná za všechno, co jsi pro mě kdy udělala! Snad ani nemusím dodávat, že máš velký podíl na tom, že se chýlím se svým doktorátem ke konci, a to ve zdraví a s úsměvem na tváři :-) Mohla bych napsat další disertačku o všech našich eskapádách a zážitcích, které jsme spolu měly možnost sdílet... Chtěla bych ti ale alespoň poděkovat za veškerou podporu, kterou jsem od tebe kdy dostala, a že jí teda bylo nepočítaně! Byla jsi se mnou v mnoha radostných, ale i hodně těžkých chvílích, a já jsem se na tebe vždycky mohla spolehnout. Takže ať už každá z nás po PhD odcestujeme 16 kamkoliv, věřím, že takové přátelství zůstane napořád :-) Jsi úžasná! Děkuju ti za všechno moje drahá, aneb “Ď”! I would also like to thank all the other groups who shared lab meetings and journal clubs with us and made them productive and interesting.